Aberrant cellular growth creates oxygen-poor microenvironments that serve to inhibit proliferation;tumors circumvent this homeostatic regulation through constitutive activation of the hypoxia response pathway, which up-regulates genes for anaerobic metabolism and angiogenesis to facilitate cellular growth that would otherwise be limited by low oxygen. The hypoxia response pathway is transcriptionally regulated by an oxygen-sensitive HIF-a (hypoxia-inducible factor-a) subunit and ARNT (aryl hydrocarbon nuclear translocator), which form a heterodimeric bHLH-PAS transcriptional activator that binds target sequences in DNA and recruits coactivators to initiate transcription. The objective of this proposal is to investigate the role of ARNT in transcriptional activation by examining the structural and functional basis of coactivator recruitment by ARNT PAS domains. We hypothesize that ARNT PAS domains simultaneously mediate interaction with HIF-a and coactivators by utilizing opposing interfaces on this small, modular domain. In Aim 1, we will identify minimal motifs for ARNT:coactivator interactions and define the molecular basis of coactivator specificity for ARNT. In Aim 2, we will determine the structural basis for the ARNT:coactivator interaction and characterize functional consequences of complex disruption in vivo. In Aim 3, we will screen ARNT PAS domains for artificial ligands to identify small molecule antagonists that disrupt the ARNT:coactivator interaction. This work will examine how ARNT contributes to transcriptional regulation in response to hypoxia and identify compounds that block ARNT function with potential therapeutic value in preventing tumor growth.